PROJECT SUMMARY Nowhere is the need for disease modifying therapeutic compounds more urgently needed than in the treatment of mental health disorders. Despite significant advances in pharmaceutical and behavior interventions, mental illnesses such as schizophrenia, depression and anxiety, combined with substance abuse disorders remain a significant and growing burden to society. Orphan G-protein coupled receptors (oGPCRs) represent an untapped reservoir of potential new drug targets for these diseases but the lack of pharmacological tools needed to validate these targets prevents their therapeutic exploitation. As a first step towards tapping this reservoir to develop new classes of medicines, we propose to find compounds that bind the oGPCRs and can be used to validate them as drug targets. Leveraging recent high-resolution gene expression maps of important brain circuits, we identified 27 oGPCRs whose discrete patterns of expression in the human brain make them high value neuroscience drug targets. These were further prioritized based on the body of biological data available for each receptor (e.g. genetics, linkage to disease) with the goal of focusing on oGPCRs of greatest relevance to mental health. For the top 10 priority targets we will conduct high-throughput screens against a library of ~50,000 compounds using an innovative ligand-induced forward trafficking assay we recently published. In this assay, each oGPCR is engineered to be retained in the endoplasmic reticulum in a state in which forward transport is enabled by the binding of a ligand. Trafficking of the oGPCR is then monitored using a split beta-galactosidase reporter system. In this way, compounds (hits) that bind and induce a conformational change in the oGPCR will be identified. Using a panel of cross and counter screen assays, hits will be confirmed and shown to engage native oGPCR. Medicinal chemistry will be used to improve potency and selectivity, and to generate chemical probes with physicochemical attributes suitable for use in cellular assays exploring the function and pharmacology of target oGPCRs. Selectivity of probes will be evaluated using a diverse panel of targets including GPCRs, ion channels, nuclear receptors and enzymes. Where a specific oGPCR is highly expressed in human iPSC-derived neuronal cultures, we will investigate the ability of probes to the specific receptor to affect neural networks using a microelectrode array assay system we developed. We anticipate that these efforts will generate >10 novel probes against high priority oGPCRs. Our intention is to rapidly distribute these to the scientific community to accelerate the understanding of these brain enriched oGPCRs, contributing to their validation as drug targets for psychological disorders.